Flow cell alignment methods and systems

ABSTRACT

A DNA flow cell processing method including positioning the flow cell on a stage at a predetermined location relative to a camera, illuminating the flow cell from a side with a first light source to reflect light off the DNA fragment bead locations, obtaining a first image of the flow cell and identifying a first reference pattern of bead locations in the first image, moving at least one of the flow cell and the stage relative to the camera, attempting to reposition the stage at the predetermined location, obtaining a second image of the flow cell, identifying the first reference pattern in the second image, and evaluating a first offset, relative to the camera, between the first reference pattern in the first image and the first reference pattern in the second image.

This application claims the benefit of and priority to U.S. applicationSer. No. 13/832,509, filed on Mar. 15, 2013, which issued as U.S. Pat.No. 9,554,095 on Jan. 24, 2017, the contents of which are incorporatedherein by reference.

BACKGROUND OF THE INVENTION

Over the past 25 years, the amount of DNA sequence information generatedand deposited into Genbank has grown exponentially. Many of thenext-generation sequencing technologies use a form of sequencing bysynthesis (SBS), wherein specially designed nucleotides and DNApolymerases are used to read the sequence of chip-bound, single-strandedDNA templates in a controlled manner. Other next-generation sequencingtechnologies may use native nucleotides and/or polymerases or labeledoligonucleotides and ligation enzymes to determine nucleic acidsequences. To attain high throughput, many millions of such templatespots, each being either single or multiple molecules, are arrayedacross a sequencing chip and their sequence is independently read outand recorded. The desire to perform high throughput sequencing stemsfrom the need for faster processing and reduced costs. However,commercial high throughput systems, while reducing the cost of largescale sequencing (e.g., 10-100 gigabases), make smaller scale sequencing(e.g., 100 megabases to 1 gigabase) costly and inconvenient.

Recently, instruments have been developed to perform sequencing on amuch smaller scale than conventional devices. Exemplary apparatus andmethods that may be used for performing smaller scale sequencingoperations are described in U.S. Patent Publication Nos. 2010/0323350(application Ser. No. 12/719,469, field Mar. 8, 2010), 2010/0152050(application Ser. No. 12/704,842, field Feb. 12, 2010), and 2009/0298131(application Ser. No. 12/370,125, field Feb. 12, 2009). The foregoingare incorporated herein by reference. Such instruments use an “assemblyline” type system, which may be arranged in the form of a carousel, tosimultaneously process a number of relatively small flow cellscontaining single-stranded DNA templates. During operation, each flowcell is physically moved through a series of processing stations. Someof these processing stations purge the flow cell and fill it with a newreagent, while others are used for imaging the flow cell, or as idlestations where the flow cell is held without substantive processing.Other processing stations may also be provided. These instrumentsprovide high throughput SBS operations, while offering significantsavings in reagents and other processing costs. This new generation ofinstruments is expected to expand the public's access to SBS operationsto use for various purposes, at a reduced cost, and with more rapidturnaround than earlier devices could offer.

There continues to be a need to advance the state of the art ofsequencing instruments, and particularly those that use movable flowcells for small-scale sequencing operations.

SUMMARY

The present invention is embodied in imagers and alignment methods foruse by imagers imaging deoxyribonucleic acid (DNA) fragments on a flowcell are disclosed. The imagers capture intensity values at DNA fragmentbead locations in tiles with each tile having a reference location inthe flow cell.

Flow cells may be aligned by obtaining a dark field image of each tileduring a first imaging session, identifying a first dark fieldconstellation of bead locations within a first tile and a second darkfield constellation of bead locations within a second tile during thefirst imaging session, identifying constellations corresponding to thefirst and second dark field constellations during a second imagingsession, altering the reference location of at least one tile during thesecond imaging session to correct for a linear offset in thecorresponding first dark field constellations, and applying at least onecorrection factor for reading out intensity values from the imager forthe bead locations in the flow cell to correct for an angular offsetdetermined from offsets in the corresponding first and second dark fieldconstellations.

An imager may include an xy stage supporting the flow cell, a cameraconfigured to capture images of flow cell tiles, and a processor coupledto the xy stage and the camera. The processor may be configured toposition the XY stage and control the camera to obtaining a dark fieldimage of each tile during a first imaging session, identify a first darkfield constellation of bead locations within a first tile and a seconddark field constellation of bead locations within a second tile duringthe first imaging session, identify constellations corresponding to thefirst and second dark field constellations during a second imagingsession, alter the reference location of at least one tile during thesecond imaging session when positioning the XY stage to correct for alinear offset in the corresponding first dark field constellations, andapply at least one correction factor for reading out intensity valuesfrom the camera for the bead locations in the flow cell to correct foran angular offset determined from offsets in the corresponding first andsecond dark field constellations.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention is best understood from the following detailed descriptionwhen read in connection with the accompanying drawings, with likeelements having the same reference numerals. When a plurality of similarelements are present, a single reference numeral may be assigned to theplurality of similar elements with a small letter designation referringto specific elements. When referring to the elements collectively or toa non-specific one or more of the elements, the small letter designationmay be dropped. Lines without arrows connecting components may representa bi-directional exchange between these components. This emphasizes thataccording to common practice, the various features of the drawings arenot drawn to scale. On the contrary, the dimensions of the variousfeatures are arbitrarily expanded or reduced for clarity. Included inthe drawings are the following figures:

FIG. 1 is a block diagram of an imager in accordance with aspects of thepresent invention;

FIG. 2 a perspective view of an imager in accordance with aspects of thepresent invention;

FIG. 3 is a side view of a flow cell within the imager of FIG. 2illustrating DNA fragment beads reflecting dark field LED light;

FIG. 4 is a top view of a flow cell within the imager of FIG. 2illustrating virtual tiles for imaging the flow cell;

FIG. 5 is a timing diagram for imaging a flow cell in accordance withaspects of the present invention;

FIG. 6 is a flow chart of steps for imaging a flow cell in accordancewith aspects of the present invention; and

FIG. 7 is a flow chart of steps for altering tile reference locationsand registering DNA fragment bead locations in accordance with aspectsof the present invention.

DETAILED DESCRIPTION OF THE INVENTION

FIG. 1 depicts an imager 100 for imaging bead locations ofdeoxyribonucleic acid (DNA) fragments on a flow cell of a chip 102 inaccordance with one embodiment of the present invention. As will bediscussed in further detail below, the imager 100 images the flow cellin tiles with each tile having a reference location in the flow cell. Atransport 104 and an xy stage 106 position the chip 102 for imaging bythe imager 100. The transport 104 moves the XY stage 106 to remove thechip from a carousel or other type of conveyor system and position thechip for imaging within the imager 100. The xy stage 106 positions thechip 102 in a horizontal plane under an objective lens 108 for imagingof individual tiles.

The imager 100 includes one or more dark field LEDs 110 and at least onebank of fluorescent LEDs 112 to illuminate the DNA fragments on the flowcell. In the illustrated embodiment, the dark field LED 110 illuminatesthe chip 102 from the side and the fluorescent field LEDs 112 illuminatethe chip 102 from above through a lens 113, an excitation filter 114,and a dichroic mirror 116. The dichroic mirror 116 reflects light in oneor more frequency bands and passes light not in those frequency band(s).The dark field LED 110 may be a red LED. The fluorescent field LEDs 112and corresponding filters 114 may be selected to illuminate the chip 102with desired colors of light.

When the dark field LEDs 110 are used to illuminate the chip, the mirror116 may be removed from the pathway such that light reflected by thebead locations in the flow cell along an imaging axis 111 passes thoughthe objective lens 108, an emission filter 109, and a lens system 109.The light then impinges upon a camera 118 where it is captured. When thefluorescent field LEDs 112 are used, light from the LEDs 112 passthrough the filter 114 and are reflected by the mirror 116 through theobjective lens 108 toward the chip 102. The bead locations emit light inresponse to being illuminated by light from the fluorescent field LEDs112 (with the emitted light having a different wavelength). The emittedlight may be produced by fluorescing dyes or other compositionsassociated with particular nucleotides, as described in U.S. applicationSer. No. 12/719,469. The emitted light passes through the objective lens108, the mirror 116, the emission filter 107, and the lens system 109 toimpinge on the camera 118. The camera may be 3296 by 2472 charge coupleddevice (CCD). Although one bank of fluorescent field LEDs 112 isillustrated, additional banks of LEDs and corresponding filters may beemployed to illuminate the chip with different colors of light. Forexample, four different colors of light may be produced, e.g., blue,green, yellow, and red.

A processor 120 is coupled to the various components to control thepositioning of the chip and the imaging of the flow cell on the chip. Amemory 122 is coupled to the processor 120. The processor 120 maycontrol the imaging system 100 to implement one or more of the stepsdescribed herein. The memory is a non-transitory computer readablemedium that may store instructions that, when carried out by theprocessor 120, implement one or more of the steps described herein.Additionally, the memory may store image information from camera 118such as intensity values at bead locations obtained during imaging.

FIG. 2 is a perspective view of the imaging system 100 depicting the xystage 106 with the chip 102 thereon in greater detail. The chip 102includes a flow cell 200 containing DNA fragments. The xy stage 106includes an x-stage 202 and corresponding controller 204 and a y-stage206 and corresponding controller 208. The processor 120 (FIG. 1) iscoupled to the x-controller 204 and the y-controller 208. The processor120 controls the x-controller 204 to move the x-stage 200 (and therebythe flow cell) along a first axis (referred to herein as the x-axis)perpendicular to an imaging axis 111. The processor 120 additionallycontrols the y-controller 208 to move the y-stage 206 (and thereby theflow cell 200) along a second axis (referred to herein as the y-axis)perpendicular to the imaging axis 111 and to the first axis.

FIG. 3 depicts a cutaway of a portion a chip 102 being illuminated bythe dark field LED 110. The flow cell 200 includes a transparent layer300 including DNA fragments thereon. The transparent layer 300 may beglass. The DNA fragments reflect light from the dark field LED 110through the objective lens 108 (FIG. 1) toward the camera 118, whichidentifies their location. These locations of DNA fragments are referredto herein as bead locations. FIG. 3 depicts four bead locations (302 a,b, c, d) although in actual use there will be many orders of magnitudemore bead locations per tile. As illustrated in FIG. 3, the dark fieldLED 110 may be tilted to optimize illumination of the beads. In anembodiment, the objective lens 108 and the dark field LED 112 arestationary in the x,y plane so that the bead illumination incidenceangle does not change.

FIG. 4 depicts a top view of a chip 102. The chip includes a flow cell200. During imaging, the flow cell 200 is imaged in tiles 400. In theillustrated embodiment, the flow cell 200 is imaged using 72 tiles(three rows of 24 tiles). Each tile has a reference locationcorresponding to the position of the tile on the chip. As will bedescribed in further detail below, at least two tiles, e.g., tile 400 aand 400 b, may be selected for use in aligning the flow cell 200 andregistering image intensities captured for individual tiles 400 withinthe flow cell 200 during imaging. The at least two tiles may be locatedessentially anywhere within the flow cell. In an embodiment, the tilesare in a center row and are at least 5 tiles away from one another,e.g., 9 tiles away from one another.

A reference constellation of bead locations within each tile, e.g., 402a and 402 b, may be used for alignment and registration purposes. In oneembodiment, one reference constellation 402 a is used to determine anoffset for the tile reference locations when the flow cell has beenremoved from the imager 100 and added back in for a subsequent imagingsession. Two or more reference constellations may be used in otherembodiments. Additionally, the difference in the offsets for twoconstellations may be used to calculate an angular adjustment for beadlocations when reading out intensity values.

FIG. 5 depicts a timing diagram 500 and FIG. 6 depicts a flow chart 600for imaging a flow cell in accordance with aspects of the presentinvention. Although the timing diagram and flow chart are described withreference to FIGS. 1-4, other suitable apparatus for carrying out thesteps described herein will be understood by one of skill in the artfrom the description herein and are considered within the scope of thepresent invention. Additionally, one or more of the steps describedherein may be omitted or performed in a different order withoutdeparting from the spirit and scope of the present invention. The totaltime for imaging all tiles in a flow cell may be on the order of 6minutes.

At step 602, a flow cell (FC) is lifted from a flow cell carrier such asa carousel. The flow cell may be lifted by a transport 104 into positionunder an objective lens 108 of an imager 100 for imaging during animaging sequence. It may take approximately 7 seconds for the transportto move the flow cell from the carrier to the imager 100 for imaging.

At block 604, the flow cell is aligned within the imager. If it is thefirst time the flow cell is positioned within the imager, a dark fieldimage of a first tile, e.g., tile 400 a, and a second tile, e.g., tile400 b, is obtained and a subset of the beads in each image is identified(referred to herein as a constellation; e.g., constellations 402 a and402 b). Thereafter, in subsequent imaging sessions, a dark field imageof the first tile and the second tile is obtained and the dark fieldimages from the current session are compared to the dark field imagesfrom the first imaging session to identify matching constellations andto determine a corresponding difference in position. The difference inposition between the constellations from at least one tile may then beused to alter the reference positions of each tile for the currentimaging session. Additionally, dark field images for all the tiles maybe obtained at this stage. It may take approximately 29 seconds or moreto align the stage and obtain the dark field images.

At block 606, a filter and mirror are positioned for fluorescent fieldimaging. The processor 102 may control the positioning of the filter 114and mirror 116, e.g., though an additional transport device (not shown).It may take approximately 4 seconds to move the filter and the mirror.

At block 608, the flow cell is positioned for imaging of a particulartile, e.g., tile 0 located in the upper left hand corner of the tiles400 (FIG. 4). The processor 102 may position the flow cell for imagingthrough the use of the x-controller 204 and the y-controller 208 (FIG.2) to move the flow cell to the reference position corresponding to thedesired tile as altered in block 604. It may take approximately 7seconds to position the flow cell at the first tile. Thereafter, it maytake a little over 0.5 second to move to the next tile to be imaged.

At block 610, the imaging system 100 autofocuses (ATF) to create a sharpimage of the flow cell tile on the camera. The processor 120 may performautofocussing by adjusting the position of the camera 118, objectivelens 108, or the chip 102 (e.g., via the transport 104 (FIG. 1)) alongthe imaging axis 111. It may take approximately 0.25 seconds to performautofocussing.

At block 612, the fluorescent LEDs are turned on. The processor 120 mayturn on the fluorescent LEDs 112. It may take approximately 120milliseconds to turn on the LEDs 112.

At block 614, the camera is triggered (snap) and, at block 616, theimaging system is synchronized. The processor 120 may trigger the camera118 to take an image and synchronize itself with the camera 118. It maytake approximately 110 milliseconds to trigger the camera 118 andapproximately 200 milliseconds to synchronize.

At block 618, the camera images the fluorescent light from the flow celltile (exposure time). The camera 118 may have an exposure period of 350milliseconds during which light from the flow cell is captured.

At block 620, an optional conversion step is performed to covert theimaged from a 1-dimensional (1D) array to a 2-dimensional (2D) array.Processor 120 may perform the conversion. This step may be performed asa programming convenience and may be omitted. It may take approximately43 milliseconds to perform the conversion.

At block 622, a decision is made regarding tiles to be processed. Ifthere are additional tiles to process, processing proceeds at block 608with the next tile and, in parallel, processing proceeds at block 624with further processing of the light captured from the current tile. Ifthere are no additional tiles, processing proceeds only at block 624.

At block 624, the image is downloaded from the camera. The processor 120may download the image from the camera 118, e.g., into random accessmemory (RAM) associated with the processor. It may take approximately700 milliseconds to download the image.

At block 626, post processing of the downloaded image is performed.Processor 120 may perform the processing and store the result in memory122. The post processing may involve registration of the captured imageswith the bead locations determined from the dark field image as adjustedbased on offsets determined at block 604. It may take approximately 365milliseconds to register the images with the bead locations andapproximately 25 milliseconds to extract the pixels, e.g., for storagein memory 122.

FIG. 7 depicts a flow chart 700 of steps describing the alignment offlow cells and the registration of captured images in further detail inaccordance with aspects of the present invention. Although flow chart700 is described with reference to FIGS. 1-4, other suitable apparatusfor carrying out the steps described herein will be understood by one ofskill in the art from the description herein and are considered withinthe scope of the present invention. Additionally, one or more of thesteps described herein may be omitted or performed in a different orderwithout departing from the spirit and scope of the present invention.

At block 702, a flow cell is positioned for a first imaging session. Aflow cell 200 on a chip 102 may be lifted by a transport 104 intoposition under an objective lens 108 of an imager 100 under control ofprocessor 120 for imaging during the first imaging session.

At block 704, a dark field image is obtained and intensity values areread out for each tile in the flow cell to be imaged. In one embodiment,a dark field image is first obtained for two reference tiles 400 withinthe flow cell 200 and a dark field image for all tiles 400 issubsequently obtained. In another embodiment, a dark field image for alltiles 400 is initially obtained without separately obtaining anotherdark field image for the two reference tiles.

The dark field image for each tile 400 may be obtained by moving theflow cell 200 to a first reference location associated with a first tileusing an xy-stage 106 under control of the processor 120, illuminatingthe side of the flow cell 200 with a dark field LED 110, capturing lightreflected by the DNA fragments with a camera 118, and storing intensityvalues in a memory 122 (with relatively high intensity valuesrepresenting the locations of the DNA fragment bead locations).Subsequent tiles within the flow cell may then be imaged by iterativelymoving the flow cell to a respective reference location associated witheach of those tiles and repeating the capturing process. During thefirst imaging session for each flow cell, the reference locations forthe tiles may be nominal locations established based on a standard flowgeometry and xy-stage 106 positions. For example, the xy-stage 106 maystart at a “home” location at one limit of the x and y travel range, andeach nominal location may be predetermined as an x and y distance fromthe home location that is expected to place the center of each tiledirectly under the objective lens 108. In this example, tile 0 may havea nominal location stored in a lookup table as (x0, y0), where x0 is adistance from the home x location, and y0 is a distance from the home ylocation, and each other tile may have its own nominal location storedin a similar manner (distances may be measured as distances, motormovement increments, and so on).

At block 706, bead locations are determined. The locations within thedark field image having a relatively high intensity value, represent thebead locations within the flow cells. The bead locations (representingthe location of the DNA fragments) may be determined by analyzing theintensity values from the dark field image with the processor 120 andselecting those with an intensity value above a value indicating thepresence of a bead. After each bead is located in the dark field, the xand y locations of the bead are stored and used in later imaging stepsas expected bead locations. For example, an array of x,y locationscorresponding to each bead may be saved for each tile. Having identifiedthe bead locations in terms of x and y locations, the full-resolutionimage itself may optionally be discarded.

At block 708, dark field reference constellations are identified withinat least two tiles. In an embodiment, the processor 120 identifies twodark field reference constellations—a first dark field referenceconstellation within a first tile and a second dark field referenceconstellation within a second tile. Each dark field referenceconstellation includes the identified bead locations located within atleast a portion of its respective tile. In an embodiment, the dark fieldreference constellation of a tile is the bead locations in anapproximately 500 by 500 pixel area in the center of the tile. The darkfield reference constellations may be determined at the time the darkfield image is obtained for the two reference tiles (i.e., in step 704),or they may be identified in a separate imaging step.

At block 710, a fluorescent field image is obtained for each tile in theflow cell to be imaged. In an embodiment, a fluorescent field image isobtained by moving the flow cell 200 to the first nominal referencelocation associated with a first tile using an xy-stage 106 undercontrol of the processor 120, illuminating the flow cell 200 with afiltered fluorescent field LED 112, and capturing light emitted by theDNA fragments with a camera 118. Subsequent tiles within the flow cellmay then be imaged by iteratively moving the flow cell to the respectivenominal reference location associated with each of those tiles, andrepeating the capturing process.

At block 712, correction factors are determined and applied. Theprocessor 120 may determine the correction factors and apply them to theexpected bead locations from the dark field image to obtain correctedbead locations.

As the xy-stage moves the flow cell for imaging the individual tiles,error may be introduced by the physical movement from tile to tile. Thiserror may be accommodated using software to compare a test region nearthe center of the fluorescent field image of each tile with the darkfield image of the same tile to determine an offset, and incorporatingthe offset into a correction factor when reading out the intensityvalues at the bead locations. The test region may be a 500 by 500 pixelarea at the center of the fluorescent field tile image. The process maybe conducted by identifying a number of x, y bead locations determinedin the dark field imaging step, and adding up the intensities of thesame locations in the 500 by 500 pixel fluorescent field test region toobtain a total intensity value. Next, the 500 by 500 pixel test regionis iteratively shifted by a predetermined amount, e.g., ±10 pixels inthe x and y directions, and the intensities at the x and y beadlocations are once again measured and summed up for each iteration.Ultimately, the test region will be shifted to a point where theintensities of the fluorescent light emitted from the beads will overliethe x,y bead locations identified in the dark field imaging step, andthe sum of the intensities will reach a peak value. At this point, theoffset is determined as the total x and y shift distances necessary toobtain the peak intensity. These x and y values are used as a correctionfactor to shift the fluorescent field tile image data and thereby alignthe intensities across the entire fluorescent field image with the beadlocations from the dark field image.

Additional correction factors may be determined. These other factors mayinclude, for example, “pincushion” correction (correcting to account fora different diffraction angle of the particular wavelength as comparedto the dark field LED), predetermined distortion corrections for knownlens aberrations, and so on.

At block 714, the intensity values of the corrected fluorescent fieldimage for the tile at the corrected bead locations are read out. Theprocessor 120 may read out the intensity values at the corrected beadlocations.

If fluorescent field images for other colors are to be determined,processing may return to block 710 with steps 710-714 repeated for eachadditional color. The flow cell is thereafter removed from the imagingstation.

At block 716, the flow cell is repositioned for a second imagingsession. A flow cell 200 on the chip 102 may be lifted by the transport104 into position under an objective lens 108 of an imager 100 forimaging during the second imaging session.

At block 718, dark field constellations corresponding to the dark fieldreference constellations identified for the flow cell during the firstimaging session (block 708) are determined. In an embodiment, theprocessor 120 identifies two dark field constellations corresponding tothe dark field referenced constellations identified during the firstimaging session—a first dark field constellation within the first tileand a second dark field constellation with a second tile. To do so, thexy-stage 106 moves the flow cell 200 to the nominal tile locations foreach of the two tiles in which the constellations should be found andtakes a dark field image of each. The dark field constellation withineach tile may be the bead locations located within a 500 by 500 pixelarea at the geometric center of the tile.

At block 720, offsets between the dark field constellations in thesecond imaging session and the dark field reference constellations inthe first imaging session are determined. The offset between theconstellations for a tile may be determined by iteratively shifting thedark field constellation in the second imaging session by apredetermined amount, e.g., ±10 pixels in the x and y direction, andevaluating the intensities of this dark field image at the x,y locationscorresponding to the bead locations found in the original referenceconstellation. The offset can be determined by summing the intensitiesof the dark field image at the expected x,y bead positions determinedfrom the dark field reference constellation at each shift iteration,with the shift iteration producing the maximum intensity at the expectedbead positions representing the offset. This may be essentially the sameprocess as described above in step 712.

In one embodiment, a single dark field constellation is compared to asingle reference constellation, and the offset between the two may beused to establish a linear offset of the flow cell in the x and/or ydirections introduced when the flow cell was repositioned for asubsequent imaging. In embodiments using a second constellation as asecond reference, the system can also calculate an angular offset thatwas introduced when the flow cell was repositioned for the subsequentimaging. For example, on each subsequent placement in the imagingstation, the new location of the first constellation 402 a may bedetermined and used as a first offset reference point, and the newlocation of the second constellation 402 b may be determined and used asa second offset reference point. By comparing the first and secondoffset reference points to the original constellation locations, thetotal x,y offset and angular rotation of the flow cell are determined.

At block 722, new reference locations for the tiles are calculated toaccommodate for any shift in the flow cell's position as compared to itsposition in the first round of imaging. In one embodiment, the processor120 uses the x,y offset from one reference constellation to calculate ageometric correction factor for each of the nominal tile centerlocations that were used in step 704. For example, if the constellationfound in step 718 was offset by +50 x pixels and +100 y pixels from thereferenced constellation location, each nominal tile center locationwould be offset a corresponding −50 x and −100 y pixels, and thexy-stage would be driven to these recalculated locations to place thecenter of each tile directly (or nearly directly) under the objectivelens for further imaging steps.

In another embodiment, the processor 120 uses the x,y offset from tworeference constellations to calculate a geometric correction factor foreach of the nominal tile center locations that were used in step 704. Inthis case, the processor 120 may calculate x, y and angular correctionfactors for each nominal tile center location to accurately position thecenter of each tile under the objective lens 108. The shape of the flowcell remains constant between successive visits to the imaging system,and therefore the calculation of the geometric correction factors is amatter of basic geometry that need not be described herein. This processis expected to be more accurate than simply using an x,y offset as inthe prior embodiment, but at the cost of greater processing time toevaluate a second constellation. As with the prior embodiment, the newreference locations are used to drive the xy-stage 106 to place thecenter of each tile directly (or nearly directly) under the objectivelens 108 for the following steps.

At block 724, fluorescent field images are obtained for each tile duringthe subsequent imaging session. The fluorescent field images may bedetermined as described above with respect to block 710 using thereference locations as altered at block 722.

At block 726, correction factor(s) are applied to the fluorescent fieldimage. The corrections factors applied in step 726 may includecorrections as described above with reference to block 712 (e.g.,registering the image to match the dark field image, pincushion anddistortion correction, etc.). In addition, a correction factor also maybe applied to rotate the fluorescent field image to correct for anyangular offset identified in step 720. Following the corrections, thefluorescent field tile image should be properly shifted in the x,ydirection and rotated to match the illuminated points and regions to thex,y locations of the beads determined from the original dark fieldimaging step for the respective tile.

At block 728, the intensity values at the corrected bead locations areread out. The processor 120 may read out the intensity values at thecorrected bead locations.

If fluorescent field images for other colors are to be determined,processing may return to block 724 with steps 724-728 repeated for eachadditional color. The flow cell is thereafter removed from the imagingstation. If the flow cell is to be imaged an additional time afterfurther processing, processing may resume at block 716.

It will be appreciated from the foregoing example that embodiments ofthe invention can be used to help resolve a significant issue relatingto sequencing by synthesis (“SBS”) instruments that use multiple flowcells that are repeatedly mounted on and dismounted from an imagingsystem. By its nature, the SBS process requires repeated examination ofexactly the same nucleotide strand to properly identify the nucleotidesas they join the sequence. In conventional systems, fixed flow cells areused. Such systems have very little free play between the flow cell andthe camera, so it is relatively easy to place the camera at almostexactly the same location during each successive imaging step. Incontrast, in systems that use flow cells that are repeatedly dismountedand mounted on the imaging platform, there is much more free play. Thefree play in such devices may derive from clearances necessary toautomate the mounting and dismounting process, manufacturing variancesbetween one flow cell and the next, and so on. In these systems, it hasbeen found that it is very difficult to properly align the flow cell inexactly the same location during successive visits to the imagingstation. Since the beads being examined may be only a few hundredmicrons in diameter (or smaller), even a small amount of misalignmentwill make it impossible to correlate the data obtained from successiveimaging steps, potentially rendering the device useless. The aboveprocess and versions thereof can be used to overcome this problem, andprovide rapid image acquisition and processing in SBS instruments thatuse removable flow cells.

Although the invention is illustrated and described herein withreference to specific embodiments, the invention is not intended to belimited to the details shown. Rather, various modifications may be madein the details within the scope and range of equivalents of the claimsand without departing from the invention.

The invention claimed is:
 1. A method for processing flow cellscontaining deoxyribonucleic acid (DNA) fragments, the method comprising:positioning a flow cell containing DNA fragment beads on an xy stagewith the flow cell facing an imaging axis of a camera, wherein the xystage is movable relative to the camera along an x-axis and a y-axis,the y-axis is perpendicular to the x-axis, and the imaging axis isperpendicular to the x-axis and the y-axis; positioning the xy stage ata first predetermined nominal location along the x-axis and the y-axiswith respect to the camera; illuminating the flow cell from a side ofand above the flow cell with a dark field light source to cause a firstlight to strike the DNA fragment beads at an angle relative to theimaging axis, and reflect off the DNA fragment bead locations; obtaininga first image of the flow cell; evaluating the first light reflectedfrom the DNA fragment beads to identify, in the first image, a firstreference pattern of bead locations; moving at least one of the flowcell and the xy stage relative to the camera; moving the xy stage toreposition the xy stage at a second position substantially at the firstpredetermined nominal location along the x-axis and the y-axis withrespect to the camera; obtaining a second image of the flow cell;identifying, in the second image, the first reference pattern of beadlocations; evaluating a first offset, relative to the camera, betweenthe first reference pattern in the first image and the first referencepattern in the second image; and using the first offset to perform afirst correction.
 2. The method of claim 1, further comprising using thefirst offset to perform a first correction comprising at least one of:moving the xy stage to reposition the flow cell relative to the camera,and applying a correction factor for reading out intensity valuesobtained during a fluorescent imaging process.
 3. The method of claim 2,wherein moving at least one of the flow cell and the xy stage relativeto the camera comprises removing the flow cell from the xy stage andreplacing the flow cell on the xy stage.
 4. The method of claim 2,wherein moving at least one of the flow cell and the xy stage relativeto the camera comprises moving the xy stage in one or both of the x-axisand the y-axis.
 5. The method of claim 2, wherein: the first offsetcomprises a linear offset between a first position of the flow cellrelative to the camera during the step of positioning the xy stage atthe first predetermined nominal location, and the second position; andperforming the first correction further comprises correcting for thelinear offset by moving the xy stage to place the flow cell at the firstposition.
 6. The method of claim 2, wherein the first offset comprises alinear offset, and applying a correction factor for reading outintensity values obtained during a fluorescent imaging process comprisescorrecting for the linear offset.
 7. The method of claim 2, wherein thefirst offset comprises an angular offset, and applying a correctionfactor for reading out intensity values obtained during a fluorescentimaging process comprises correcting for the angular offset.
 8. Themethod of claim 2, further comprising: positioning the xy stage at asecond predetermined nominal location along the x-axis and the y-axiswith respect to the camera; obtaining a third image of the flow cell;evaluating the first light reflected from the DNA fragment beads toidentify, in the third image, a second reference pattern of beadlocations; after the step of moving at least one of the flow cell andthe xy stage relative to the camera, moving the xy stage to repositionthe xy stage at a fourth position along the x-axis and the y-axis withrespect to the camera; obtaining a fourth image of the flow cell;identifying, in the fourth image, the second reference pattern of beadlocations; and evaluating a second offset, relative to the camera,between the second reference pattern in the third image and the secondreference pattern in the fourth image.
 9. The method of claim 8, furthercomprising using the second offset to perform a second correctioncomprising at least one of: moving the xy stage to reposition the flowcell relative to the camera, and applying a second correction factor forreading out intensity values obtained during the fluorescent imagingprocess.
 10. The method of claim 9, wherein the first correctioncomprises a linear correction and the second correction comprises anangular correction.
 11. The method of claim 8, wherein the first offsetcomprises a linear offset, and the second offset comprises an angularoffset.
 12. The method of claim 8, wherein the first offset comprises afirst linear offset, the second offset comprises a second linear offset,and the method further comprises: evaluating the first linear offset andthe second linear offset to determine an angular offset.
 13. The methodof claim 12, further comprising using the angular offset to perform asecond correction comprising at least one of: moving the xy stage toreposition the flow cell relative to the camera, and applying an angularcorrection factor for reading out intensity values obtained during thefluorescent imaging process.
 14. The method of claim 8, wherein: theflow cell comprises a plurality of tiles, each tile having a respectivereference location; the first image and the second image are made at afirst tile; and the third image and the fourth image are made at asecond tile.
 15. The method of claim 14, wherein the second tile isspaced from the first tile.
 16. The method of claim 15, wherein thefirst offset comprises a first linear offset, the second offsetcomprises a second linear offset, and the method further comprises:evaluating the first linear offset and the second linear offset todetermine an angular offset.
 17. The method of claim 16, furthercomprising using the angular offset to perform a second correctioncomprising at least one of: moving the xy stage to reposition the flowcell relative to the camera, and applying an angular correction factorfor reading out intensity values obtained during the fluorescent imagingprocess.
 18. The method of claim 1, further comprising: obtaining athird image of the flow cell; evaluating the first light reflected fromthe DNA fragment beads to identify, in the third image, a secondreference pattern of bead locations; obtaining a fourth image of theflow cell; identifying, in the fourth image, the second referencepattern of bead locations; and evaluating a second offset, relative tothe camera, between the second reference pattern in the third image andthe second reference pattern in the fourth image.
 19. The method ofclaim 18, further comprising evaluating the first offset and the secondoffset to determine an angular offset.
 20. The method of claim 19,further comprising: using the first offset to perform a first correctioncomprising at least one of: moving the xy stage to reposition the flowcell relative to the camera, and applying a correction factor forreading out intensity values obtained during a fluorescent imagingprocess; and using the angular offset to perform a second correctioncomprising at least one of: moving the xy stage to reposition the flowcell relative to the camera, and applying an angular correction factorfor reading out intensity values obtained during a fluorescent imagingprocess.
 21. The method of claim 18, further comprising: using the firstoffset to perform a first linear correction comprising at least one of:moving the xy stage to reposition the flow cell relative to the camera,and applying a linear correction factor for reading out intensity valuesobtained during a fluorescent imaging process; and using the secondoffset to perform an angular correction comprising applying an angularcorrection factor for reading out intensity values obtained during afluorescent imaging process.